On each experimental occasion, C. coli ATCC® 43478™ (LGC Standards GmbH, Wesel, Germany), previously stored at −80°C in nutrient broth No. 2 (Oxoid, Basingstoke, England) supplemented with 5% lysed horse blood (Oxoid) and 20% glycerol (BDH VWR, Lutterworth, England), was recovered on Columbia blood agar (CBA; bioMérieux, Marcy-l’Étoile, France), then isolated on modified charcoal cefoperazone deoxycholate agar (CCDA; Oxoid) and subcultured once more on CBA. Plate incubation was always performed at 41.5 ± 1°C for 24 h under microaerobic conditions (GENbox microaer and GENbox jar, bioMérieux). For the generation of pure C. coli suspensions, one pure colony from CBA was inoculated in 100 ml of cation-adjusted Mueller-Hinton broth (CAMHB; BBL™ BD, Franklin Lakes, NJ) following incubation at 37 ± 1°C for 20 ± 2 h under microaerobic conditions. The growth state of the bacterial culture was approximately estimated at the end of the incubation by measuring the optical density (OD) at 600 nm using a spectrophotometer (BioPhotometer®; Eppendorf, Hamburg, Germany). The estimated count was confirmed by subjecting three aliquots of 100 ml to qPCR, as described below, and by performing 10-fold serial dilutions in buffered peptone water (BPW, LabM, United Kingdom) followed by spread plating on double CBA plates.
Modified charcoal cefoperazone deoxycholate agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, France
Modified charcoal-cefoperazone deoxycholate agar (mCCDA) is a culture medium used for the selective isolation and identification of Campylobacter species from food and environmental samples. The agar contains charcoal, cefoperazone, and deoxycholate, which inhibit the growth of competing microorganisms and facilitate the isolation of Campylobacter.
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Lab products found in correlation
Campygen, by Thermo Fisher Scientific (2 mentions)
Columbia agar, by Thermo Fisher Scientific (2 mentions)
Lysed horse blood, by Thermo Fisher Scientific (1 mentions)
Nutrient broth no 2, by Thermo Fisher Scientific (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Genbox microaer, by bioMérieux (1 mentions)
Genbox jar, by bioMérieux (1 mentions)
Buffered peptone water (bpw), by Lab M (1 mentions)
Hippurate hydrolysis test, by Thermo Fisher Scientific (1 mentions)
Columbia blood agar, by Thermo Fisher Scientific (1 mentions)
Vitek ms system, by bioMérieux (1 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (1 mentions)
Nutrient broth, by Thermo Fisher Scientific (1 mentions)
Sr0155, by Thermo Fisher Scientific (1 mentions)
Campygen 2.5 l sachet, by Thermo Fisher Scientific (1 mentions)
Campygentm, by Thermo Fisher Scientific (1 mentions)
Mueller hinton agar (mha), by BD (1 mentions)
Sr155e, by Thermo Fisher Scientific (1 mentions)
Preston campylobacter selective enrichment broth, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
13 protocols using modified charcoal cefoperazone deoxycholate agar
1
Campylobacter coli Cultivation Protocol
2
Enumeration and Speciation of Campylobacter
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Enumeration of Campylobacter in samples was performed according to ISO/TS 10272-2:2017. As required by the ISO method, tenfold dilutions of samples on BPW were performed. Then, 0.1 mL from each inoculum was plated onto mCCDA (Modified Charcoal Cefoperazone Deoxycholate Agar, Oxoid, Dardilly, France) and incubated at 41.5 ± 1 °C in a micro-aerobic atmosphere (84% N2, 10% CO2, 6% O2) for 44 h ± 4 h. For further confirmation analysis, five Campylobacter-like colonies were purified on blood agar (AES La-boratories®, Bruz Cedex, France) at 41.5 ± 1 °C in a microaerobic atmosphere (84% N2, 10% CO2, 6% O2) over 24 h. Then, colony morphology and motility under dark field microscopy were evaluated. Confirmation of presumptive Campylobacter colonies was assessed by oxidase and catalase tests and plating at different temperatures and atmospheres (25 °C under aerobic conditions and 41.5 °C under microaerophilic conditions for 24 h) in Columbia blood agar (AES Laboratories).
For Campylobacter speciation, isolated strains were plated on Columbia blood agar (Oxoid Ltd., Basingstoke, UK) and incubated at 41.5 °C for 44 ± 4 h in a modified atmosphere (5% O2, 85% N2, 10% CO2, CampyGen, Oxoid, Basingstoke, UK). Finally, the hippurate hydrolysis test (Oxoid, Madrid, Spain) was used to determine the species of the Campylobacter [38 (link),42 (link)].
For Campylobacter speciation, isolated strains were plated on Columbia blood agar (Oxoid Ltd., Basingstoke, UK) and incubated at 41.5 °C for 44 ± 4 h in a modified atmosphere (5% O2, 85% N2, 10% CO2, CampyGen, Oxoid, Basingstoke, UK). Finally, the hippurate hydrolysis test (Oxoid, Madrid, Spain) was used to determine the species of the Campylobacter [38 (link),42 (link)].
3
Isolation and Identification of Acinetobacter spp. from Meat Samples
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Acinetobacter spp. were isolated as previously described in the Korean Food Code 2020 for Campylobacter spp. isolation [8 ]. Twenty-five grams of each meat sample was inoculated into 225ml of Bolton medium (Oxoid, UK) and cultured at 42°C under microaerophilic conditions (5% O2, 85% N2, and 10% CO2) for 48h. Next, the cultures were spread on modified Charcoal-Cefoperazone-Deoxycholate Agar (Oxoid) and incubated at 42°C under microaerophilic conditions for 24h. Subsequently, three or more colonies with a circular or irregular shape and translucent or transparent in appearance in grayish-white tones were selected and identified using the VITEK-MS system (BioMérieux Vitek, France). The isolates, identified as belonging to “A. baumannii complex,” were selected for further molecular characterization.
4
Isolation and characterization of Campylobacter from chicken
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The Campylobacter isolates used in this study were isolated during a separate study investigating the efficacy of sanitizers during chicken meat processing (Chousalkar et al., 2019 (link)). Briefly, chicken meat carcasses (15 birds from each of eight different broiler production sheds) were collected from two separate processing plants prior to the inside-outside wash step. Bacteria were isolated by massaging chicken carcasses (prior to sanitizer exposure) in buffered peptone water (BPW) (Oxoid, Australia). Two hundred microliters of BPW wash was spread plated onto modified charcoal-cefoperazone deoxycholate agar (mCCDA) (Oxoid, Australia) and incubated at 42°C in 10% CO2 for 48 h. Putative, Campylobacter isolates were sub-cultured once to obtain pure cultures, stored at −80°C in 5% glycerol, and further characterized using PCR. A total of 116 Campylobacter isolates were obtained.
In preparation for experiments, bacteria were resuscitated from freezing stocks on to Columbia sheep blood agar (SBA) (Oxoid, Thermo Scientific, Australia) and incubated at 42°C in 10% CO2 for 48 h. The Campylobacter jejuni ATCC 33291 strain was used as a control strain.
In preparation for experiments, bacteria were resuscitated from freezing stocks on to Columbia sheep blood agar (SBA) (Oxoid, Thermo Scientific, Australia) and incubated at 42°C in 10% CO2 for 48 h. The Campylobacter jejuni ATCC 33291 strain was used as a control strain.
5
Quantifying Campylobacter Contamination on Chicken Skin
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To examine the level of Campylobacter spp. contamination on the skin of
chicken carcasses, approximately 10-g samples of breast and back skin were removed from
three different areas (total 6 pieces) with sterilized scalpel and forceps, and
transferred to a sterilized stomaching bag. The skin samples were stomached for 1 min in
90 ml of Preston enrichment medium containing nutrient broth (Oxoid, Basingstoke, UK) with
Preston Campylobacter selective supplement SR0117 (Oxoid) and 5%
defibrinated horse whole blood (Nippon Bio-Sipp Center, Tokyo, Japan). Serial ten-fold
dilutions of these suspensions were made in Preston enrichment medium. After
microaerophilic culture at 42°C for 48 hr (under mixed gas: 80% N2, 10%
CO2, 5% O2, and 5% H2), 10 µl of the culture solution
in each tube for the most probable number (MPN) was inoculated onto modified Charcoal
Cefoperazone Deoxycholate Agar (mCCDA; Oxoid) supplemented with CCDA selective supplement
SR0155 (Oxoid), then incubated again under the same microaerophilic conditions as those
described above. The number of campylobacters was obtained by applying the common 3-tubes
MPN procedure at each dilution [30 (link)].
chicken carcasses, approximately 10-g samples of breast and back skin were removed from
three different areas (total 6 pieces) with sterilized scalpel and forceps, and
transferred to a sterilized stomaching bag. The skin samples were stomached for 1 min in
90 ml of Preston enrichment medium containing nutrient broth (Oxoid, Basingstoke, UK) with
Preston Campylobacter selective supplement SR0117 (Oxoid) and 5%
defibrinated horse whole blood (Nippon Bio-Sipp Center, Tokyo, Japan). Serial ten-fold
dilutions of these suspensions were made in Preston enrichment medium. After
microaerophilic culture at 42°C for 48 hr (under mixed gas: 80% N2, 10%
CO2, 5% O2, and 5% H2), 10 µl of the culture solution
in each tube for the most probable number (MPN) was inoculated onto modified Charcoal
Cefoperazone Deoxycholate Agar (mCCDA; Oxoid) supplemented with CCDA selective supplement
SR0155 (Oxoid), then incubated again under the same microaerophilic conditions as those
described above. The number of campylobacters was obtained by applying the common 3-tubes
MPN procedure at each dilution [30 (link)].
6
Campylobacter Detection from Stool Samples
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Stool specimens were cultured for Campylobacter using modified ISO methods (EN ISO 10272-2019) for detecting and enumerating Campylobacter [54 (link)] by direct plating and by filtration of stool prior to plating. For the direct plating, a 10 μl aliquot of each stool specimen was directly plated onto modified charcoal-cefoperazone deoxycholate agar (mCCDA) with cefoperazone and amphotericin B supplements (Oxoid, Hampshire, United Kingdom). In parallel, a 4 ml aliquot of each stool specimen was emulsified in 4 ml of phosphate-buffered saline (PBS) and filtered through a 0.65 μm syringe filter (Sartorius, Göttingen, Germany), before 10 µl was inoculated onto a mCCDA plate. All plates throughout the protocol were incubated in a microaerophilic atmosphere using anaerobic jars with CampyGen 2.5 L sachet (Oxoid, Hampshire, United Kingdom) at 37 °C for 48 h. C. jejuni strain 81116 was used as a positive control throughout the protocol.
Once incubated, up to 30 suspected Campylobacter colonies per patient specimen were sub-cultured onto mCCDA for purification, and further sub-cultured onto Columbia agar with 5% horse blood (Oxoid, Hampshire, United Kingdom). Colony morphology, microscopy, and oxidase test (Thermo Fisher Scientific, Loughborough, United Kingdom) were utilised to confirm presumptive Campylobacter isolates.
Once incubated, up to 30 suspected Campylobacter colonies per patient specimen were sub-cultured onto mCCDA for purification, and further sub-cultured onto Columbia agar with 5% horse blood (Oxoid, Hampshire, United Kingdom). Colony morphology, microscopy, and oxidase test (Thermo Fisher Scientific, Loughborough, United Kingdom) were utilised to confirm presumptive Campylobacter isolates.
7
Campylobacter Detection in Animal Faeces
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Faecal samples from each animal were obtained from the rectum and transferred to 100 ml plastic jars. A new and clean pair of gloves were used for each sample and the jars were filled to a maximum of two‐thirds, in order to decrease the risk of the lid opening during transport. The samples were transported chilled to the National Veterinary Institute (SVA, Uppsala, Sweden). All packages reached the laboratory within 48 h. The samples were analysed by direct culture according to ISO 10272: Part 1C (2017). In brief, faecal contents were spread on modified charcoal‐cefoperazone‐deoxycholate agar (mCCDA) (Oxoid, Basingstoke, UK) and the plates were incubated at 37·0°C for 44 ± 4 h in microaerobic atmosphere generated by the use of CampyGenTM (Oxoid). Identification of Campylobacter spp. was based on typical morphological aspects, white to grey colonies with metallic sheen and phase‐contrast microscopic observation with corkscrew movement according to ISO 10272: part 1 (2017).
8
Isolation of Campylobacter jejuni and C. coli from Poultry and Environment
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The C. jejuni and C. coli strains used in this study were isolated from turkey feces or ceca, from swine feces, or from the environment at different farms in eastern North Carolina, South Carolina, or Virginia between 2003 and 2014 (Table 1 ). Strains were first isolated using direct plating on modified charcoal cefoperazone deoxycholate agar (mCCDA; Oxoid, Basingstoke, Hampshire, England) and incubated in anaerobic jars containing a CampyPak Plus microaerobic system (Becton Dickinson, Sparks, MD) at 42°C for 48h (Smith et al., 2004 (link)). Strains were then subcultured on different media: (i) non-selective Mueller-Hinton agar (MHA; Becton Dickinson, United States), (ii) modified selective Campy Line agar (CLA-S; Line, 2001 (link)) containing 25μg/ml sulfamethoxazole in addition to the original antibiotics (trimethoprim, vancomycin, and polymyxin B; Line et al., 2008 (link)), and (iii) selective and differential CHROMagar™ Campylobacter (CAC; CHROMagar, Paris, France), and incubated at 42°C in an 10% CO2 incubator.
9
Campylobacter Isolation from Meat Samples
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The meats were put onto modified charcoal cefoperazone deoxycholate agar (mCCDA) (Oxoid Ltd.) including the Campylobacter mCCDA‐selective supplement, SR155E (Oxoid Ltd.) following arrival at the laboratory. CampyGenTM gas packets were used to create microaerophilic situations, which were maintained at 37°C for 48 h (Oxoid). Campylobacter colonies that are grayish, flat, wet, and spread easily were subcultured on Mueller Hinton Agar enriched with 5% defibrinated horse blood (MHS) and cultured at 37°C for 48 h under microaerophilic conditions. Isolates of Campylobacter were stored at 80°C in Mueller Hinton broth containing 25% glycerol (v/v).
10
Campylobacter Species Isolation Protocol
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Isolation of Campylobacter species was performed under microaerobic conditions according to the protocol established by Vandepitte et al. [18 ]. Samples were enriched in Preston Campylobacter selective enrichment broth (Oxoid, Cambridge, UK) at 42 °C for 48 h. The enrichment broth was plated onto modified charcoal cefoperazone deoxycholate agar (mCCDA; Oxoid, Cambridge, UK) then transferred onto Columbia agar (Oxoid, Cambridge, UK) plates supplemented with 5% sterile defibrinated horse blood. Presumptive Campylobacter colonies were confirmed by oxidase, catalase, hippurate, and indoxyl acetate hydrolyses biochemical tests, in addition to testing their susceptibilities to nalidixic acid and cephalothin antimicrobials (30 mg/disc, each) [19 ]. The bacterial DNA was extracted using a QIAamp DNA Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplifications of the 23S rRNA gene of Campylobacter species [20 (link)] in addition to mapA and ceuE genes of C. jejuni and C. coli, respectively, [21 (link)] were applied using oligonucleotide primers listed in Table S1 .
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